Eukaryotic cells utilize small membrane-bound vesicles to transport cargo between subcellular organelles, and to the plasma membrane for secretion. The proper function and specificity of the vesicular transport and membrane fusion processes are crucial for maintenance of cellular integrity, growth, cellular movement and secretory events such as hormone release and neurotransmission. Vesicle transport and fusion at the plasma membrane require many essential proteins, including the SNARE proteins and Sec1p that are involved in the membrane fusion process, the Rab and Rho GTPases, and a large complex called the exocyst. The exocyst complex has been implicated in a number of different functions: selection of the site of exocytosis;physical tethering of secretory vesicles to these sites;communicating with cytoskeletal and cell cycle proteins;and regulating the specificity and assembly of the SNARE proteins. None of these are well understood at the molecular level. Our aim is to combine biochemical and biophysical techniques with genetics and cell biological methods in order to understand the molecular functions of the exocyst complex. We have chosen to study the exocyst proteins from the model organism Saccharomyces cerevisiae so as to take advantage of the wealth of genetic and cell biological techniques available. To accomplish this goal, we are i) investigating the protein- protein interactions within the exocyst complex through biochemical and biophysical studies in vitro and analyzing the function of the exocyst in vivo through characterization of specific mutants;(ii) characterizing the role of the exocyst in SNARE complex assembly;and (iii) identifying and characterizing several novel regulators of the exocyst and SNARE complex assembly. Because these proteins are conserved from yeast to human neurons, this research will advance our knowledge of how secretion and growth are regulated in all eukaryotic cells. PUBLIC HEALTH RELEVANCE: Life depends upon proper cellular growth and development, from simple processes such as cell growth and division of unicellular eukaryotes, to very complicated interactions between the neurons in the brain. Cellular growth, development, movement and communication absolutely require proper vesicle targeting and membrane fusion. An essential component of these fundamental cell biological processes is the exocyst complex, which is conserved from yeast to man. Our studies of the exocyst and its regulation of the membrane fusion proteins will lead to a molecular understanding of the function of the yeast exocyst complex. In addition, our research will also lead to the development of many constructs, reagents and ideas that will be valuable tools for studying how the exocyst complex regulates secretion in all eukaryotic cells.